NR4A1 destabilizes TNF mRNA in microglia and modulates stroke outcomes

Microglia play a dual role in stroke depending on their pro-inflammatory and anti-inflammatory polarization. A study in PLOS Biology identifies a new mechanism, through which the transcription factor NR4A1 negatively regulates TNF expression in microglia.

Funding: This work was partially supported by NIH grants (RF1AG065345, R21AG073862, R21AG064422, and R01HL146574) to YY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests:
The authors have declared that no competing interests exist. macrophages and T cells, the authors further generate mutant mice with NR4A1 deficiency in microglia only using the microglia-specific TMEM119-CreER T2 line. Similar to the NR4A1 global knockout mice, loss of NR4A1 in microglia specifically results in increased TNF expression and aggravated outcomes after ischemic stroke. These findings strongly indicate that microglial NR4A1 regulates TNF expression and stroke pathogenesis.
The work by Liu and colleagues is novel and intriguing as it identifies a previously unidentified role of NR4A1 as an RNA-binding protein, highlights a new regulatory mechanism of TNF expression in microglia, and underscores a key function of NR4A1 in microglial polarization and stroke outcomes. These findings provide new molecular targets with therapeutic potential. For example, NR4A1 may be up-regulated to decrease the production of pro-inflammatory cytokines in microglia and attenuate their pro-inflammatory polarization. This will likely ameliorate neuroinflammation and improve neurological symptoms in stroke and neurodegenerative disorders. Since pro-inflammatory microglia play an important role in pathogen clearance, NR4A1 up-regulation may aggravate injury in infectious diseases. In addition, the timing of NR4A1 activation may also affect disease progression and outcomes. Therefore, it is important to investigate the therapeutic effects of NR4A1 manipulation in various neurological diseases and at multiple time points.
Next, whether NR4A1 uses a similar mechanism to regulate the expression of other proinflammatory cytokines needs to be validated in vivo. Although both in vitro and in vivo studies support the hypothesis that NR4A1 negatively regulates TNF expression in microglia, evidence supporting NR4A1's inhibitory role in the expression of other pro-inflammatory cytokines (e.g., IL-1β and IL-6) is only found in vitro. How to reconcile the different results of IL-1β and IL-6 expression in vitro and in vivo? Based on the observation that up-regulated NR4A1 is mainly found in microglial cytoplasm in vivo but in both cytoplasm and nucleus in vitro, the authors speculate that different pro-inflammatory cytokines are regulated by NR4A1 through distinct mechanisms. Specifically, cytoplasmic NR4A1 regulates TNF expression posttranscriptionally via binding to and destabilizing TNF mRNA, while nuclear NR4A1 regulates the expression of IL-1β and IL-6 at the transcriptional level as a transcription factor. This hypothesis is further confirmed by expressing NR4A1 exclusively in the cytoplasm. What confers the different subcellular distribution of NR4A1 in vitro and in vivo, however, remains unknown and should be explored in the future. This information will allow fine-tuning of neuroinflammation, which will likely enhance the efficacy and reduce the side effects of potential therapies.
In addition, it remains unclear which domain of NR4A1 binds to m 6 A sites and how exactly it interacts with m 6 A. Identifying the specific domains that bind to m 6 A-modified mRNAs and understanding the dynamics of such interaction will provide insights into the molecular mechanism of microglial activation and promote the development of novel treatments for various neurological diseases.
Finally, the translational potential of this study should be validated using human microglial cells in the future. Many studies report species-specific features of microglia [10,11]. Therefore, findings in rodents need to be validated in human cells. Although this study [9] confirmed upregulation of NR4A1 in microglia after stroke in postmortem human brains, it is unclear whether NR4A1 modulates TNF expression via the same posttranscriptional mechanism in human microglia. Validating this observation in human microglia or microglia derived from induced pluripotent stem cells would significantly increase the translational potential of this work.